Underivatized, aqueous soluable β(1-3) glucan, composition and method of making same

ABSTRACT

The present invention relates to neutral, aqueous soluble β-glucans which exert potent and specific immunological effects without stimulating the production of certain cytokines, to preparations containing the novel β-glucans, and to a novel manufacturing process therefor. The neutral, aqueous soluble β-glucan preparation has a high affinity for the β-glucan receptor of human monocytes and retains two primary biological (or immunological) activities, (1) the enhancement of microbicidal activity of phagocytic cells, and (2) monocyte, neutrophil and platelet hemopoietic activity. Unlike soluble glucans described in the prior art, the neutral, aqueous soluble β-glucan of this invention neither induces nor primes IL-1β and TNFα production in vitro and in vivo. Safe and efficacious preparations of neutral, aqueous soluble β-glucan of the present invention can be used in therapeutic and/or prophylactic treatment regimens of humans and animals to enhance their immune response, without stimulating the production of certain biochemical mediators (e.g., IL-1β, TNFα) that can cause detrimental side effects, such as fever and inflammation.

This application is a continuation of co-pending application Ser. No.08/373,251 filed Jan. 26, 1995, which is U. S. National Phase ofPCT/US93/07904, filed Aug. 20, 1993, which is a Continuation-in-Part ofU.S. application Ser. No. 07/934,015, filed Aug. 21, 1992, now U.S. Pat.No. 5,622,939.

BACKGROUND OF THE INVENTION

In the early 1960's, zymosan, a crude insoluble yeast extract preparedby boiling yeast before and after trypsin treatment, was noted toproduce marked hyperplasia and functional stimulation of thereticuloendothelial system in rodents. In animal studies, zymosanpreparations were shown to inactivate complement component C3, toenhance antibody formation, to promote survival following irradiation,to increase resistance to bacterial infections, to inhibit tumordevelopment, to promote graft rejection, and to inhibit dietary-inducedhypercholesterolemia and cholesterosis. Zymosan was shown to consist ofpolysaccharides, proteins, fats, and inorganic elements; however,subsequent studies identified the active components of the yeast cellwall as a pure polysaccharide, specifically β-glucan. In conventionalnomenclature, the polysaccharide β-glucan is known aspoly-(1-6)-β-D-glucopyranosyl-(1-3)-β-D-glucopyranose (PGG). Repetitionof biological assays with β-glucan indicated that most of the abovefunctional activities identified with zymosan were retained by thepurified β-glucan preparation.

The properties of β-glucan are quite similar to those of endotoxin inincreasing nonspecific immunity and resistance to infection. Theactivities of β-glucan as an immune adjuvant and hemopoietic stimulatorcompare to those of more complex biological response modifiers (BRMs),such as bacillus Calmette-Guerin (BCG) and Corynebacterium Rarvum. Thefunctional activities of yeast β-glucan are also comparable to thosestructurally similar carbohydrate polymers isolated from fungi andplants. These higher molecular weight (1-3)-β-D-glucans such asschizophyllan, lentinan, krestin, grifolan, and pachyman exhibit similarimmunomodulatory activities. A common mechanism shared by all theseβ-glucan preparations is their stimulation of cytokines such asinterleukin-1(IL-1) and tumor necrosis factor (TNF). Lentinan has beenextensively investigated for its antitumor properties, both in animalmodels at 1 mg/kg for 10 days and in clinical trials since the late1970's in Japan for advanced or recurrent malignant lymphoma andcolorectal, mammary, lung and gastric cancers. In cancer chemotherapy,lentinan has been administered at 0.5-5 mg/day, intramuscularly (I.M.)or intravenously (I.V.), two or three times per week alone, or incombination with antineoplastic drugs. In addition to the activitiesascribed to yeast glucans, studies suggest lentinan acts as a T-cellimmunopotentiator, inducing cytotoxic activities, including productionof interleukins 1 and 3 and colony-stimulating factors (CSF). (Chiharaet al., 1989, Int. J. Immunotherapy, 4:145-154; Hamuro and Chihara, InLentinan , An Immunorotentiator)

Various preparations of both particulate and soluble β-glucans have beentested in animal models to evaluate biological activities. The use ofsoluble and insoluble β-glucans alone or as vaccine adjuvants for viraland bacterial antigens has been shown in animal models to markedlyincrease resistance to a variety of bacterial, fungal, protozoan andviral infections. The hemopoietic effects of β-glucan have beencorrelated with increased peripheral blood leukocyte counts and bonemarrow and splenic cellularity, reflecting increased numbers ofgranulocyte-macrophage progenitor cells, splenic pluripotent stem cells,and erythroid progenitor cells, as well as, increased serum levels ofgranulocyte-monocyte colony-stimulating factor (GM-CSF). Furthermore,the hemopoietic and anti-infective effects of β-glucan were active incyclophosphamide-treated immunosuppressed animals. β-glucan was shown tobe beneficial in animal models of trauma, wound healing andtumorigenesis. However, various insoluble and soluble preparations ofβ-glucan differed significantly in biological specificity and potency,with effective dosages varying from 25to 500 mg/kg intravenously orintraperitoneally (I.P.) in models for protection against infection andfor hemopoiesis. Insoluble preparations demonstrated undesirabletoxicological properties manifested by hepatosplenomegaly and granulomaformation. Clinical interest was focused on a soluble glucan preparationwhich would retain biological activity yet yield negligible toxicitywhen administered systemically. Chronic systemic administration of asoluble phosphorylated glucan over a wide range of doses (40-1000 mg/kg)yielded negligible toxicity in animals (DiLuzio et al., 1979, Int. J. ofCancer, 24:773-779; DiLuzio, U.S. Pat. No. 4,739,046).

The molecular mechanism of action of β-glucan has been elucidated by thedemonstration of specific β-glucan receptor binding sites on the cellmembranes of human neutrophils and macrophages. Mannans, galactans,α(1-4)-linked glucose polymers and β(1-4)-linked glucose polymers haveno avidity for this receptor. These β-glucan binding sites areopsonin-independent phagocytic receptors for particulate activators ofthe alternate complement pathway, similar to Escherichia colilipopolysaccharide (LPS) and some animal red blood cells. Ligand bindingto the β-glucan receptor, in the absence of antibody, results incomplement activation, phagocytosis, lysosomal enzyme release, andprostaglandin, thromboxane and leukotriene generation; therebyincreasing nonspecific resistance to infection. However, solubleβ-glucan preparations described in the prior art demonstratedstimulation of cytokines. Increases in plasma and splenic levels ofinterleukins 1 and 2 (IL-1, IL-2) in addition to TNF were observed invivo and corresponded to induction of the synthesis of these cytokinesin vitro. (See Sherwood et a l., 1987, Int. J. Immunopharmac., 9:261-267(enhancement of IL-1 and IL-2 levels in rats injected with solubleglucan); Williams et al., 1988, Int. J. Immuno pharmac., 10:405-414(systemic administration of soluble glucan to AIDS patients increasedIL-1and IL-2levels which were accompanied by chills and fever); Browderet al., 1990, Ann. Sura., 211:605-613 (glucan administration to traumapatients increased serum IL-1 levels, but not TNF levels); Adachi etal., 1990, Chem. Pharm. Bull., 38:988-992 (chemically cross-linkedβ(1-3) glucans induced IL-1 production in mice).)

Interleukin-1 is a primary immunologic mediator involved in cellulardefense mechanisms. Numerous studies have been carried out on theapplication of IL-1 to enhance non-specific resistance to infection in avariety of clinical states. Pomposelli et al., J. Parent. Ent. Nutr.12(2):212-218, (1988). The major problem associated with the excessivestimulation or exogenous administration of IL-1 and other cellularmediators in humans is toxicity and side effects resulting from thedisruption of the gentle balance of the immunoregulatory network. Fauciet al., Ann. Int. Med., 106:421-433 (1987). IL-1 is an inflammatorycytokine that has been shown to adversely affect a variety of tissuesand organs. For instance, recombinant IL-1 has been shown to causedeath, hypotensive shock, leukopenia, thrombocytopenia, anemia andlactic acidosis. In addition, IL-1 induces sodium excretion, anorexia,slow wave sleep, bone resorption, decreased pain threshold andexpression of many inflammatory-associated cytokines. It is also toxicto the insulin secreting beta cells of the pancreas. Patients sufferingfrom a number of inflammatory diseases have elevated levels of IL-1 intheir systems. Administration of agents that enhance furtherIL-1production only exacerbate these inflammatory conditions.

Tumor necrosis factor is also involved in infection, inflammation andcancer. Small amounts of TNF release growth factors while in largeramounts, TNF can cause septic shock, aches, pains, fever, clotting ofblood, degradation of bone and stimulation of white blood cells andother immune defenses.

SUMMARY OF THE INVENTION

The present invention relates to neutral soluble β-glucans which enhancea host's immune defense mechanisms to infection but do not induce aninflammatory response, to preparations containing the neutral solubleβ-glucans, and to a novel manufacturing process therefor. In the presentmethod, soluble glucan which induces cytokine production is processedthrough a unique series of acid, alkaline and neutral treatment steps toyield a conformationally pure neutral soluble glucan preparation withunique biological properties. The neutral soluble glucan preparationretains a specific subset of immunological properties common toβ-glucans but uniquely does not induce the production of IL-1 and TNF invitro or in vivo. Throughout this specification, unless otherwiseindicated, the expressions "neutral soluble glucan" and "neutral solubleglucan" refer to the composition prepared as described in Example 1.

The neutral soluble glucan preparation is produced by treating insolubleglucan with acid to produce a water soluble glucan, dissociating thenative conformations of the soluble glucan at alkaline pH, purifying thedesired molecular weight fraction at alkaline pH, re-annealing thedissociated glucan fraction under controlled conditions of time,temperature and pH to form a unique triple helical conformation, andfurther purifying under neutral pH to remove single helix and aggregatedmaterials to yield a conformationally pure, neutral, water soluble,underivatized glucan which has a unique biological profile.

The neutral soluble glucan preparation has a high affinity for theβ-glucan receptor of human monocytes and retains two primary biologicalactivities, (1) the enhancement of microbicidal activity of phagocyticcells, and (2) monocyte, neutrophil and platelet hemopoietic activity.Unlike soluble glucans described in the prior art, the neutral solubleglucan of this invention neither induces nor primes mononuclear cells toincrease IL-1 and TNF production in vitro and in vivo.

The neutral soluble glucan preparation is appropriate for parenteral(e.g., intravenous, intraperitoneal, subcutaneous, intramuscular),topical, oral or intranasal administration to humans and animals as ananti-infective to combat infection associated with burns, surgery,chemotherapy, bone marrow disorders and other conditions in which theimmune system may be compromised. Neutral soluble glucan produced by thepresent method can be maintained in a clear solution and equilibrated ina pharmaceutically acceptable carrier. Safe and efficacious preparationsof the neutral soluble glucan of the present invention can be used intherapeutic and/or prophylactic treatment regimens of humans and animalsto enhance their immune response, without stimulating the production ofcertain biochemical mediators (e.g., IL-1 and TNF) that can causedetrimental side effects, such as fever and inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the general structure of neutral soluble glucan as being alinear β(1-3)-linked glucose polymer having periodic branching via asingle β(1-6)-linked glucose moiety.

FIG. 2 shows a gel permeation chromatogram (pH 7) of soluble glucanwhich has not been purified by alkali dissociation and re-annealing. Thechromatogram shows three species, referred to herein as high molecularweight aggregate (Ag), Peak A and Peak B (single helix glucan).

FIG. 3 is a chromatogram obtained for the neutral soluble glucan by gelpermeation chromatography. The solid line represents the neutral solubleglucan at pH 7 and the broken line represents the neutral soluble glucanat pH 13.

FIG. 4 is a chromatogram obtained for the single helix β-glucan (Peak B)by gel permeation chromatography. The solid line represents Peak B at pH7 and the broken line represents Peak B at pH 13.

FIG. 5 shows the change in serum TNF levels, over time, taken frompatients intravenously infused with placebo (broken line) or neutralsoluble glucan (solid line).

FIG. 6 shows the change in serum IL-1 levels, over time, taken frompatients intravenously infused with placebo (broken line) or neutralsoluble glucan (solid line).

FIG. 7 is a diagram representing peripheral blood counts from irradiatedmice following administration of neutral soluble glucan.

FIG. 8 is a diagram representing platelet cell counts fromcisplatin-treated mice following administration of neutral solubleglucan.

DETAILED DESCRIPTION OF INVENTION

The invention relates to a neutral soluble β-glucan polymer that canbind to the β-glucan receptor and activate only a desired subset ofimmune responses. The terms "neutral soluble β-glucan" and "neutralsoluble glucan", unless otherwise specified, refer to the compositionprepared as described in Example 1.

This neutral soluble β-glucan has been shown to increase the number ofneutrophils and monocytes as well as their direct infection fightingactivity (phagocytosis and microbial killing). However, the neutralsoluble β-glucan does not stimulate the production of biochemicalmediators, such as IL-1 and TNF, that can cause detrimental side effectssuch as high fever, inflammation, wasting disease and organ failure.These advantageous properties make neutral soluble glucan preparationsof this invention useful in the prevention and treatment of infectionbecause they selectively activate only those components of the immunesystem responsible for the initial response to infection, withoutstimulating the release of certain biochemical mediators that can causeadverse side effects. The solution containing the neutral solubleβ-glucan also lacks the toxicity common to many immunomodulators.

The neutral soluble β-glucans of this invention are composed of glucosemonomers organized as a β(1-3) linked glucopyranose backbone withperiodic branching via β(1-6) glycosidic linkages. The neutral solubleglucan preparations contain glucans, which have not been substantiallymodified by substitution with functional (e.g., charged) groups or othercovalent attachments. The general structure of the neutral solubleglucan is shown in FIG. 1. The biologically active preparation of thisinvention is a conformationally purified form of β-glucan produced bydissociating the native glucan conformations and re-annealing andpurifying the resulting unique triple helical conformation. The uniqueconformation of the neutral soluble glucan contributes to the glucan'sability to selectively activate the immune system without stimulatingthe production of detrimental biochemical mediators.

The neutral soluble glucan preparations of this invention are preparedfrom insoluble glucan particles, preferably derived from yeastorganisms. See Manners et al., Biochem. J., 135:19-30, (1973) for ageneral procedure to make insoluble yeast glucans. Glucan particleswhich are particularly useful as starting materials in the presentinvention are whole glucan particles (WGP) described by Jamas et al., inU.S. Pat. Nos. 4,810,646, 4,992,540, 5,082,936 and 5,028,703, theteachings of all of which are hereby incorporated herein by reference.The source of the whole glucan particles can be the broad spectrum ofglucan-containing yeast organisms which contain β-glucans in their cellwalls. Whole glucan particles obtained from the strains Saccharomycescerevisiae R4 (NRRL Y-15903; deposit made in connection with U.S. Pat.No. 4,810,646) and R4 Ad (ATCC No. 74181) are particularly useful. Otherstrains of yeast that can be used include Saccharomyces delbrueckii,Saccharomyces rosei, Saccharomyces microellipsodes, Saccharomycescarlsberaensis, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces fragilis, Kluyveromyces polys porus, Candida albicans,Candida cloacae, Candida tropicalis, Candida utilis, Hansenula wingei,Hansenula arni, Hansenula henricii, Hansenula americana.

A procedure for extraction of whole glucan particles is described byJamas et al., in U.S. Pat. Nos. 4,810,646, 4,992,540, 5,082,936 and5,028,703. For the purpose of this present invention, it is notnecessary to conduct the final organic extraction and wash stepsdescribed by Jamas et al.

In the present process, whole glucan particles are suspended in an acidsolution under conditions sufficient to dissolve the acid-soluble glucanportion. For most glucans, an acid solution having a pH of from about 1to about 5 and at a temperature of from about 20° to about 100° C. issufficient. Preferably, the acid used is an organic acid capable ofdissolving the acid-soluble glucan portion. Acetic acid, atconcentrations of from about 0.1 to about 5M or formic acid atconcentrations of from about 50% to 98% (w/v) are useful for thispurpose. The treatment time may vary from about 10 minutes to about 20hours depending on the acid concentration, temperature and source ofwhole glucan particles. For example, modified glucans having more β(1-6)branching than naturally-occurring, or wild-type glucans, require morestringent conditions, i.e., longer exposure times and highertemperatures. This acid-treatment step can be repeated under similar orvariable conditions. One preferred processing method is described in theexemplification using glucan derived from S. cerevisiae strain R4 Ad. Inanother embodiment of the present method, whole glucan particles fromthe strain, S. cerevisiae R4, which have a higher level of β(1-6)branching than naturally-occurring glucans, are used, and treatment iscarried out with 90% (w/v) formic acid at 20° C. for about 20 minutesand then at 85° C. for about 30 minutes.

The insoluble glucan particles are then separated from the solution byan appropriate separation technique, for example, by centrifugation orfiltration. The pH of the resulting slurry is adjusted with an alkalinecompound such as sodium hydroxide, to a pH of about 7 to about 14. Theprecipitate is collected by centrifugation and is boiled in purifiedwater (e.g., USP) for three hours. The slurry is then resuspended in hotalkali having a concentration sufficient to solubilize the glucanpolymers. Alkaline compounds which can be used in this step includealkali-metal or alkali-earth metal hydroxides, such as sodium hydroxideor potassium hydroxide, having a concentration of from about 0.01 toabout 10N. This step can be conducted at a temperature of from about 4°C. to about 121° C., preferably from about 20° C. to about 100° C. Inone embodiment of the process, the conditions utilized are a 1M solutionof sodium hydroxide at a temperature of about 80°-100° C. and a contacttime of approximately 1-2 hours. The resulting mixture containssolubilized glucan molecules and particulate glucan residue andgenerally has a dark brown color due to oxidation of contaminatingproteins and sugars. The particulate residue is removed from the mixtureby an appropriate separation technique, e.g., centrifugation and/orfiltration. In another embodiment of the process the acid-solubleglucans are precipitated after the preceding acid hydrolysis reaction bythe addition of about 1.5 volumes of ethanol. The mixture is chilled toabout 4° C. for two (2) hours and the resulting precipitate is collectedby centrifugation or filtration and washed with water. The pellet isthen resuspended in water, and stirred for three (3) to twelve (12)hours at a temperature between about 20° C. and 100° C. At this pointthe pH is adjusted to approximately 10 to 13 with a base such as sodiumhydroxide.

The resulting solution contains dissociated soluble glucan molecules.This solution is now purified to remove traces of insoluble glucan andhigh molecular weight soluble glucans which can cause aggregation. Thisstep can be carried out by an appropriate purification technique, forexample, by ultrafiltration, utilizing membranes with nominal molecularweight (NMW) levels or cut-offs in the range of about 1,000 to 100,000daltons. It was discovered that in order to prevent gradual aggregationor precipitation of the glucan polymers the preferred membrane for thisstep has a nominal molecular weight cut-off of about 100,000 daltons.The soluble glucan is then further purified at alkaline pH to remove lowmolecular weight materials. This step can be carried out by anappropriate purification technique, for example, by ultrafiltration,utilizing membranes with nominal molecular weight levels or cut-offs inthe range of 1,000 to 30,000 daltons.

The resulting dissociated soluble glucan is re-annealed under controlledconditions of time (e.g., from about 10 to about 120 minutes),temperature (e.g., from about 50° to about 70° C.) and pH. The pH of thesolution is adjusted to the range of about 3.5-11 (preferably 6-8) withan acid, such as hydrochloric acid. The purpose of this re-annealingstep is to cause the soluble glucan to rearrange from a single helixconformation to a new ordered triple helical conformation. There-annealed glucan solution is then size fractionated, for example byusing 30,000-70,000 NMW and 100,000-500,000 NMW cut-off membraneultrafilters to selectively remove high and low molecular weight solubleglucans. Prior to sizing, the soluble glucans exist as a mixture ofconformations including random coils, gel matrices or aggregates, triplehelices and single helices. The objective of the sizing step is toobtain an enriched fraction for the re-annealed conformation of specificmolecular weight. The order in which the ultrafilters are used is amatter of preference.

The concentrated fraction obtained is enriched in the soluble,biologically active neutral soluble glucan. The glucan concentrate isfurther purified, for example, by diafiltration using a 10,000 daltonmembrane. The preferred concentration of the soluble glucan after thisstep is from about 2 to about 10 mg/ml.

The neutralized solution can then be further purified, for example, bydiafiltration, using a pharmaceutically acceptable medium (e.g., sterilewater for injection, phosphate-buffered saline (PBS), isotonic saline,dextrose) suitable for parenteral administration. The preferred membranefor this diafiltration step has a nominal molecular weight cut-off ofabout 10,000 daltons. The final concentration of the glucan solution isadjusted in the range of about 0.5 to 10 mg/ml. In accordance withpharmaceutical manufacturing standards for parenteral products, thesolution can be terminally sterilized by filtration through a 0.22 μmfilter. The neutral soluble glucan preparation obtained by this processis sterile, non-antigenic, essentially pyr bgen-free, and can be storedat room temperature (e.g., 15°-30° C.) for extended periods of timewithout degradation. This process is unique in that it results in aneutral aqueous solution of (pH 4.5 to 7.0) immunologically activeglucans which is suitable for parenteral administration.

For purposes of the present invention, the term "soluble" as used hereinto describe glucans obtained by the present process, means a visuallyclear solution can be formed in an aqueous medium such as water, PBS,isotonic saline, or a dextrose solution having a neutral pH (e.g., fromabout pH 5 to about 7.5), at room temperature (about 20°-25° C.) and ata concentration of up to about 10 mg/ml. The term "aqueous medium"refers to water and water-rich phases, particularly to pharmaceuticallyacceptable aqueous liquids, including PBS, saline and dextrosesolutions. The expression "visually clear" means that at a concentrationof 1 mg/ml, the absorption of the solution at 530 nm is less than OD0.01 greater than the OD of an otherwise identical solution lacking theB-glucan component.

The resulting solution is substantially free of protein contamination,is non-antigenic, non-pyrogenic and is pharmaceutically acceptable forparenteral administration to animals and humans. However, if desired,the soluble glucan can be dried by an appropriate drying method, such aslyophilization, and stored in dry form.

The neutral soluble glucans of this invention can be used as safe,effective, therapeutic and/or prophylactic agents, either alone or asadjuvants, to enhance the immune response in humans and animals. Solubleglucans produced by the present method selectively activate only thosecomponents that are responsible for the initial response to infection,without stimulating or priming the immune system to release certainbiochemical mediators (e.g., IL-1, TNF, IL-6, IL-8 and GM-CSF) that cancause adverse side effects. As such, the present soluble glucancomposition can be used to prevent or treat infectious diseases inmalnourished patients, patients undergoing surgery and bone marrowtransplants, patients undergoing chemotherapy or radiotherapy,neutropenic patients, HIV-infected patients, trauma patients, burnpatients, patients with chronic or resistant infections such as thoseresulting from myelodysplastic syndrome, and the elderly, all of who mayhave weakened immune systems. An immunocompromised individual isgenerally defined as a person who exhibits an attenuated or reducedability to mount a normal cellular and/or humoral defense to challengeby infectious agents, e.g., viruses, bacteria, fungi and protozoa. Aprotein malnourished individual is generally defined as a person who hasa serum albumin level of less than about 3.2 grams per deciliter (g/dl)and/or unintentional weight loss of greater than 10% of usual bodyweight.

More particularly, the method of the invention can be used totherapeutically or prophylactically treat animals or humans who are at aheightened risk of infection due to imminent surgery, injury, illness,radiation or chemotherapy, or other condition which deleteriouslyaffects the immune system. The method is useful to treat patients whohave a disease or disorder which causes the normal metabolic immuneresponse to be reduced or depressed, such as HIV infection (AIDS). Forexample, the method can be used to pre-initiate the metabolic immuneresponse in patients who are undergoing chemotherapy or radiationtherapy, or who are at a heightened risk for developing secondaryinfections or post-operative complications because of a disease,disorder or treatment resulting in a reduced ability to mobilize thebody's normal metabolic responses to infection. Treatment with theneutral soluble glucans has been shown to be particularly effective inmobilizing the host's normal immune defenses, thereby engendering ameasure of protection from infection in the treated host.

The present composition is generally administered to an animal or ahuman in an amount sufficient to produce immune system enhancement. Themode of administration of the neutral soluble glucan can be oral,enteral, parenteral, intravenous, subcutaneous, intraperitoneal,intramuscular, topical or intranasal. The form in which the compositionwill be administered (e.g., powder, tablet, capsule, solution, emulsion)will depend upon the route by which it is administered. The quantity ofthe composition to be administered will be determined on an individualbasis, and will be based at least in part on consideration of theseverity of infection or injury in the patient, the patient's conditionor overall health, the patient's weight and the time available beforesurgery, chemotherapy or other high-risk treatment. In general, a singledose will preferably contain approximately 0.01 to approximately 10 mgof modified glucan per kilogram of body weight, and preferably fromabout 0.1 to 2.5 mg/kg. The dosage for topical application will dependupon the particular wound to be treated, the degree of infection andseverity of the wound. A typical dosage for wounds will be from about0.001 mg/ml to about 2 mg/ml, and preferably from about 0.01 to about0.5 mg/ml.

In general, the compositions of the present invention can beadministered to an individual periodically as necessary to stimulate theindividual's immune response. An individual skilled in the medical artswill be able to determine the length of time during which thecomposition is administered and the dosage, depending upon the physicalcondition of the patient and the disease or disorder being treated. Asstated above, the composition may also be used as a preventativetreatment to preinitiate the normal metabolic defenses which the bodymobilizes against infections.

Neutral soluble β-glucan can be used for the prevention and treatment ofinfections caused by a broad spectrum of bacterial, fungal, viral andprotozoan pathogens. The prophylactic administration of neutral solubleβ-glucan to a person undergoing surgery, either preoperatively,intraoperatively and/or post-operatively, will reduce the incidence andseverity of post-operative infections in both normal and high-riskpatients. For example, in patients undergoing surgical procedures thatare classified as contaminated or potentially contaminated (e.g.,gastrointestinal surgery, hysterectomy, cesarean section, transurethralprostatectomy) and in patients in whom infection at the operative sitewould present a serious risk (e.g., prosthetic arthroplasty,cardiovascular surgery), concurrent initial therapy with an appropriateantibacterial agent and the present neutral soluble glucan preparationwill reduce the incidence and severity of infectious complications.

In patients who are immunosuppressed, not only by disease (e.g., cancer,AIDS) but by courses of chemotherapy and/or radiotherapy, theprophylactic administration of the soluble glucan will reduce theincidence of infections caused by a broad spectrum of opportunisticpathogens including many unusual bacteria, fungi and viruses. Therapyusing neutral soluble β-glucan has demonstrated a significantradio-protective effect with its ability to enhance and prolongmacrophage function and regeneration and, as a result enhance resistanceto microbial invasion and infection.

In high risk patients (e.g., over age 65, diabetics, patients havingcancer, malnutrition, renal disease, emphysema, dehydration, restrictedmobility, etc.) hospitalization frequently is associated with a highincidence of serious nosocomial infection. Treatment with neutralsoluble β-glucan may be started empirically before catheterization, useof respirators, drainage tubes, intensive care units, prolongedhospitalizations, etc. to help prevent the infections that are commonlyassociated with these procedures. Concurrent therapy with antimicrobialagents and the neutral soluble β-glucan is indicated for the treatmentof chronic, severe, refractory, complex and difficult to treatinfections.

The compositions administered in the method of the present invention canoptionally include other components, in addition to the neutral solubleβ-glucan. The other components that can be included in a particularcomposition are determined primarily by the manner in which thecomposition is to be administered. For example, a composition to beadministered orally in tablet form can include, in addition to neutralsoluble β-glucan, a filler (e.g., lactose), a binder (e.g.,carboxymethyl cellulose, gum arabic, gelatin), an adjuvant, a flavoringagent, a coloring agent and a coating material (e.g., wax orplasticizer). A composition to be administered in liquid form caninclude neutral soluble β-glucan and, optionally, an emulsifying agent,a flavoring agent and/or a coloring agent. A composition for parenteraladministration can be mixed, dissolved or emulsified in water, sterilesaline, PBS, dextrose or other biologically acceptable carrier. Acomposition for topical administration can be formulated into a gel,ointment, lotion, cream or other form in which the composition iscapable of coating the site to be treated, e.g., wound site.

Compositions comprising neutral soluble glucan can also be administeredtopically to a wound site to stimulate and enhance wound healing andrepair. Wounds due to ulcers, acne, viral infections, fungal infectionsor periodontal disease, among others, can be treated according to themethods of this invention to accelerate the healing process.Alternatively, the neutral soluble β-glucan can be injected into thewound or afflicted area. In addition to wound repair, the compositioncan be used to treat infection associated therewith or the causativeagents that result in the wound. A composition for topicaladministration can be formulated into a gel, ointment, lotion, cream orother form in which the composition is capable of coating the site to betreated, e.g., wound site. The dosage for topical application willdepend upon the particular wound to be treated, the degree of infectionand severity of the wound. A typical dosage for wounds will be fromabout 0.01 mg/ml to about 2 mg/ml, and preferably from about 0.01 toabout 0.5 mg/ml.

Another particular use of the compositions of this invention is for thetreatment of myelodysplastic syndrome (MDS). MDS, frequently referred toas preleukemia syndrome, is a group of clonal hematopoietic stem celldisorders characterized by abnormal bone marrow differentiation andmaturation leading to peripheral cytopenia with high probability ofeventual leukemic conversion. Recurrent infection, hemorrhaging andterminal infection resulting in death typically accompany MDS. Thus, inorder to reduce the severity of the disease and the frequency ofinfection, compositions comprising modified glucan can be chronicallyadministered to a patient diagnosed as having MDS according to themethods of this invention, in order to specifically increase theinfection fighting activity of the patient's white blood cells. Otherbone marrow disorders, such as aplastic anemia (a condition ofquantitatively reduced and defective hematopoiesis) can be treated toreduce infection and hemorrhage that are associated with this diseasestate.

Neutral soluble glucan produced by the present method enhances thenon-specific defenses of mammalian mononuclear cells and significantlyincreases their ability to respond to an infectious challenge. Theunique property of neutral soluble glucan macrophage activation is thatit does not result in increased body temperatures (i.e., fever) as hasbeen reported with many non-specific stimulants of those defenses. Thiscritical advantage of neutral soluble glucan may lie in the naturalprofile of responses it mediates in white blood cells. It has been shownthat the neutral soluble β-glucan of the present invention selectivelyactivates immune responses but does not directly stimulate or primecytokine (e.g., IL-1 and TNF) release from mononuclear cells, thusdistinguishing the present neutral soluble glucan from other glucanpreparations (e.g., lentinan, krestin) and immunostimulants.

In addition, it has been demonstrated herein that the neutral solubleglucan preparation of the present invention possesses an unexpectedplatelet stimulating property. Although it was known that glucans havethe ability to stimulate white blood cell hematopoiesis, the disclosedplatelet stimulating property had not been reported or anticipated. Thisproperty can be exploited in a therapeutic regimen for use as anadjuvant in parallel with radiation or chemotherapy treatment. Radiationand chemotherapy are known to result in neutropenia (reducedpolymorphonuclear (PMN) leukocyte cell count) and thrombocytopenia(reduced platelet count). At present, these conditions are treated bythe administration of colony-stimulating factors such as GM-CSF andgranulocyte colony-stimulating factor (G-CSF). Such factors areeffective in overcoming neutropenia, but fail to impact uponthrombocytopenia. Thus, the platelet stimulating property of the neutralsoluble glucan preparation of this invention can be used, for example,as a therapeutic agent to prevent or minimize the development ofthrombocytopenia which limits the dose of the radiation orchemotherapeutic agent which is used to treat cancer.

The invention is further illustrated by the following Examples.

EXAMPLES Example 1

Preparation of Neutral Soluble Glucan From S. Cerevisiae

Saccharomyces cerevisiae strain R4 Ad (a non-recombinant derivative ofwild-type strain A364A), was grown in a large-scale fermentation cultureusing a defined glucose, ammonium sulfate minimal medium. The productionculture was maintained under glucose limitation in a feed-batch mode(New Brunswick MPP80). When the growing culture reached late logarithmicphase, the fermentation was ended and the βglucan was stabilized byadjusting the culture to pH 12±0.5 using 10M NaOH. The yeast cellscontaining β-glucan were harvested by continuous-flow centrifugation(Westfalia SA-1). After centrifugation, the cells were collected into astainless steel extraction vessel.

The first step in the extraction process was an alkaline extractionaccomplished by mixing the cells with 1M sodium hydroxide (NaOH) at90°±5°0 C. for 1 hour. Upon completion of this alkaline extraction, theβ-glucan remained in the solid phase, which was collected by continuouscentrifugation (Westfalia SA-1). The collected cell wall fraction wasextracted a second time using the same procedure and under the sameconditions. Treatment with alkali hydrolyzed and solubilized thecellular proteins, nucleic acids, mannans, soluble glucans and polarlipids into the supernatant fraction, and deacetylated chitin tochitosan in the cell wall.

The second step in the extraction process was a pH 4.5±0.05 (adjustedwith concentrated HCl) extraction at 75°±5° C. for 1 hour. This wasfollowed by a 0.1M acetic acid extraction to complete the removal ofglycogen, chitin, chitosan and remaining proteins. The solids werecollected and rinsed twice with Purified Water USP to remove anyresidual acid as well as any yeast degradation products.

The third step in the extraction process was a set of six organicextractions. The first four extractions were carried out in isopropanol.The solids were collected by centrifugation and then subjected to twoacetone extractions. The two-stage organic extractions eliminatednonpolar lipids and hydrophobic proteins which may have co-purified withthe drug substance. The resulting wet solids were dried in a vacuum ovenat 65°±5° C. for 48-96 hours to yield a free-flowing powder.

At this stage the extraction process yielded a stable, insolubleintermediate consisting of approximately 90% β-glucan, called wholeglucan particles (WGPs). The dry WGP intermediate was stored at 15°-30°C. until further use.

The WGP powder was resuspended in 98% (w/v) formic acid, in a glassreaction vessel at room temperature. The resulting mixture was heated to85°±5° C. for 20 minutes. Under these conditions, the WGPs werepartially hydrolyzed and solubilized to provide the desired molecularweight distribution of soluble β-glucan which was then precipitated byadding 1.5 volumes of ethanol. After complete mixing, the preparationwas centrifuged to collect the β-glucan precipitate. Any residual formicacid was removed by boiling the βglucan preparation in Purified WaterUSP for three hours.

Any unhydrolyzed WGPs were then removed from the β-glucan solution bycentrifugation. The β-glucan solution was raised to pH 12.5±0.5 by theaddition of the concentrated sodium hydroxide. The remainingpurification steps were carried out by ultrafiltration.

The soluble alkaline β-glucan preparation was passed through a 100,000nominal molecular weight (NMW) cut-off membrane ultrafilter (AmiconDC10). Under alkaline conditions this membrane ultrafilter removedinsoluble and high molecular weight soluble β-glucan. Trace lowmolecular weight degradation products were then removed by recirculationthrough a 10,000 NMW cut-off membrane ultrafilter. The ultrafiltrationwas conducted as a constant volume wash with 0.1 M NaOH.

The βglucan solution was re-annealed under controlled conditions byadjusting the pH to 7.0±0.5 with concentrated hydrochloric acid, heatingto 60°±10° C., which was maintained for 20 minutes and then cooled. Theneutral re-annealed solution was then concentrated and washed withSodium Chloride Injection USP in a 70,000 NMW cut-off membraneultrafilter (Filtron Minisep) to enrich for the re-annealed neutralsoluble glucan. Next the material was filtered through a 300,000 NMWcut-off membrane ultrafilter (Filtron Minisep) to remove high molecularweight and aggregated glucan molecules. In the same ultrafilter, theneutral soluble glucan material was washed with Sodium ChlorideInjection USP in a constant volume wash mode.

The neutral soluble glucan was then concentrated in a 10,000 NMW cut-offmembrane ultrafilter. The concentration process continued until aconcentration of at least 1.0 mg/ml hexose equivalent was achieved.

The resulting neutral soluble glucan was then subjected to filtrationthrough a depyrogenating filter (0.1 micron Posidyne) and a sterile 0.2micron filter (Millipak) to yield sterile, pyrogen-free neutral solubleglucan. The neutral soluble glucan solution was stored at controlledroom temperature (15°-30° C.) until further use. The aqueous solubilityof neutral soluble glucan in the pH range of 4 to 8 is approximately 100mg/ml. The solubility increased with increasing pH and reached approx.150 mg/ml at pH 13.

Example 2

Analysis of Neutral Soluble Glucan

A. Glucose, Mannose and Glucosamine

Monosaccharide analysis was performed to quantitate the relative amountsof β-glucan (as glucose), mannan or phosphomannan (as mannose), andchitin (as N-acetyl glucosamine) in the neutral soluble glucan. Thesample was hydrolyzed to monosaccharides in 2 M trifluoroacetic acid for4 hours at 110° C., evaporated to dryness, and redissolved in water.Monosaccharides were separated on a Dionex HPLC system using a CarboPacPA100 column (4×250 mm) using 5M NaOH at 1 ml/min and quantitated usinga pulsed electrochemical detector (Dionex Model PED-1). The sensitivityof this assay for monosaccharides is 0.1% (w/w).

Glucose (retention time of 16.6 min) was identified as the onlymonosaccharide component of neutral soluble glucan along with traces ofglucose degradation products (from hydrolysis) anhydroglucose at 2.5 minand 5-hydroxymethylfurfural at 4.3 min. The results confirm that neutralsoluble glucan consisted of ≧98% glucose.

B. FTIR

Fourier transform infrared spectroscopy by diffuse reflectance (FTIR,Matson Instruments, Polaris) of lyophilized neutral soluble glucansamples was used to determine the anomeric structure (α vs. β), andlinkage type (β(1-3), β(1-6), β(1-4)) present in neutral soluble glucan.Absorption maxima of 890 cm⁻¹ identified β(1-3) linkages; 920 cm⁻¹identified β(1-6) linkages. No presence of α-linked anomers (e.g.,glycogen, 850 cm⁻) or β(1-4)-linked polysaccharides (e.g., chitin, 930cm⁻¹) were detected.

Example 3

Conformational Analysis

A solution of β-glucan which was not processed by alkali dissociationand re-annealing was analyzed for its compositional identity by gelpermeation chromatography (pH 7) and found to contain multiple species,referred to herein as high molecular weight aggregate (Ag), Peak A andPeak B (See FIG. 2). Neutral soluble glucan which was prepared by alkalidissociation and re-annealing as described in Example 1, is present as asingle peak (see FIG. 3) with an average molecular weight of 92,660daltons at pH 7. The distinct conformations of neutral soluble glucanand Peak B were demonstrated by gel permeation chromatography at pH 7and pH 13 using a refractive index detector. Neutral soluble glucanunderwent a significant conformational transition from pH 7 to pH 13which illustrates complete dissociation of the multiple helix at pH 7 toa single helical form at pH 13 (see FIG. 3). In contrast, Peak B onlyunderwent a slight shift in molecular weight from pH 7 to pH 13 (seeFIG. 4). The molecular weight of neutral soluble glucan and Peak Bglucans as a function of pH is shown below in Table 1.

                  TABLE 1                                                         ______________________________________                                                     MW         MW Ratio                                              Sample        pH 7      pH 13   (pH 7/pH 13)                                  ______________________________________                                        Neutral soluble glucan                                                                      92,666    18,693  4.96                                          Peak B         8,317     7,168  1.16                                          ______________________________________                                    

The conformation of neutral soluble glucan and Peak B glucan was alsodetermined by aniline blue complexing (Evans et al., 1984, Carb. Pol.,4:215-230; Adachi et al., 1988, Carb. Res., 177:91-100), using curdlan,a linear β(1-3) glucan, as the triple helix control and pustulan, aβ(1-6) glucan, as a non-ordered conformational control. The results arediscussed below and shown in Table 2.

The curdlan triple helix control complexed with aniline blue resultingin high fluorescence. Increasing the NaOH concentration began todissociate the curdlan triple helix slightly, but NaOHconcentrations >0.25M are required for complete dissociation of curdlan.The pustulan non-ordered control only formed a weak complex with anilineblue resulting in low fluorescence measurements which were not affectedby NaOH concentration.

The neutral soluble glucan complexed effectively with aniline blue atlow NaOH concentration (25 mM NaOH) resulting in high fluorescence.However, the neutral soluble glucan conformation dissociatedsignificantly (50%) at NaOH concentrations as low as 150 mM NaOHindicating that it exists as a unique conformation compared to naturallyoccurring β-glucans, such as laminarin and curdlan, which requiresignificantly higher NaOH concentrations for dissociation to occur. PeakB formed a weak complex with aniline blue due to its single helicalconformation.

                  TABLE 2                                                         ______________________________________                                        Conformational Analysis of Glucans by Aniline Blue Complexing                             Fluorescence                                                                    25 mM      100 mM   150 mM                                      Test Material NaOH       NaOH     NaOH                                        ______________________________________                                        Blank         0          2        0                                           Curdlan       53.5       41.6     36                                          β(1-3) glucan                                                            Pustulan      9.8        8.3      8.0                                         β(1-6) glucan                                                            Neutral soluble glucan                                                                      40         25.6     20.2                                        Peak B        12.4       6.2      4.1                                         ______________________________________                                    

Example 4

Effects of Neutral Soluble Glucan on Human Monocyte Production ofTNF.alpha.

Human peripheral blood mononuclear cells were isolated (Janusz et al.,(1987), J. Immunol., 138: 3897-3901) from normal citrated anddextran-treated blood, washed in Hank's balanced salt solution (HBSS),lacking calcium, magnesium, and phenol red, and purified by gradientcentrifugation on cushions of Ficoll-Paque (Pharmacia Fine Chemicals,Piscataway, N.J.). The mononuclear cells were collected into HBSS,washed twice, resuspended in RPMI 1640 Medium (Gibco, Grand Island,N.Y.) containing 1% heat-inactivated autologous serum (56° C. for 30min.), and counted on the Coulter counter.

For the preparation of monocyte monolayers, 1 ml of 2.2×10⁶ mononuclearcells/ml was plated into wells of 24-well tissue culture plates (CoStar,Cambridge, Mass.), incubated for 1 hour at 37° C. in a humidifiedatmosphere of 5% CO₂, and washed three times with RPMI to removenonadherent cells. A second 1 ml aliquot of 2.2×10⁶ mononuclear cells/mlwas layered into each well and incubated for 2 hours described abovebefore removal of the nonadherent cells. By visual enumeration at 40×with an inverted phase microscope and a calibrated reticle, the numberof adherent cells for 30 different donors was 0.77±0.20×10⁶ per well(mean ±SD). By morphology and nonspecific esterase staining, >95% of theadherent cells were monocytes.

Monocyte monolayers were incubated at 37° C. in the CO₂ chamber for 0 to8 hours with 0.5 ml of RPMI, 1% heat-inactivated autologous serum, 10 mMHEPES, and 5 mM MgCl₂ in the absence and presence of various glucanpreparations. The culture supernatant was removed, clarified bycentrifugation at 14,000 g for 5 min at 4° C., and stored at -70° C.before assay of TNFα.

The concentration of TNFα in the monocyte supernatants was measured byan enzyme-linked immunoadsorbent assay (ELISA) with the BIOKINE TNF Testkit (T Cell Sciences, Cambridge, Mass.), which had a lower limit ofdetectability of 40 pg/ml. The data are expressed as pg per 10⁶monocytes, which was calculated by dividing the quantity of cytokine in0.5 ml of supernatant by the number of monocytes per well.

For the determination of cell-associated levels of TNFα, the adherentmonocytes were lysed in 0.25 ml PBS by three rounds of freezing andthawing, the lysates were cleared of debris by centrifugation at 14,000g for 5 min at 4° C., and the resulting supernatants were stored at -70°C. Newly prepared monocyte monolayers contained no detectable levels ofintracellular TNFα.

The results are shown in Tables 3 and 4 below.

                  TABLE 3                                                         ______________________________________                                        TNFα Synthesis by Human Monocytes                                       Stimulated with Various Glucan Preparations                                                   TNFα                                                                    (pg/10.sup.6 monocytes)                                       Glucan       Conc.    1      2    3    Mean ± SD                           ______________________________________                                        Buffer Control         36     39    2   26 ± 21                            Neutral soluble glucan                                                                     1 mg/ml   44     51   33  43 ± 9                              Laminarin    1 mg/ml   372    324  227 308 ± 74                            Whole Glucan particles                                                                     4 × 10.sup.7 /ml                                                                 2129   1478 1683 1763 ± 333                          ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        TNFα Stimulation by                                                     Different Conformational Structures of Soluble β-Glucan                                           TNFα                                           Glucan          Conc.    (pg/10.sup.6 monocytes)                              ______________________________________                                        Buffer Control  1 mg/ml   40                                                  Laminarin       1 mg/ml  1312                                                 Neutral soluble glucan                                                                        1 mg/ml   16                                                  Peak B          1 mg/ml  1341                                                 Glucan particles                                                                              4 × 10.sup.7 /ml                                                                 2065                                                 ______________________________________                                    

Table 3 shows that TNFα was stimulated by insoluble glucan particles andby laminarin, a soluble β(1-6) and β(1-3) linked glucan. There was nostimulation of TNFα by neutral soluble glucan. Table 4 shows similarresults, but further confirms that TNFα stimulation is dependent uponconformational structure. The neutral soluble glucan did not stimulateTNFα while Peak B (single helical conformation) did stimulate TNFα.

Example 5

Avidity of Neutral Soluble Glucan for the Glucan Receptor

Monolayers of human monocytes, prepared on siliconized glass coverslips(Czop et al., 1978, J. Immunol., 120:1132), were incubated for 18minutes at 37° C. in a humidified 5% CO₂ incubator with either 0.25 mlof buffer (RPMI-Mg-HEPES) or a range of concentrations (0.1-50 μg/ml) ofneutral soluble glucan. The monocyte monolayers were then washed twicewith 50 ml of RPMI 1640 medium and were layered with 0.25 ml of 4.8×10⁶/ml zymosan particles (Czop and Austen, 1985, J. Immunol.,134:2588-2593). After a 30 minute incubation at 37° C., the monolayerswere washed three times with 50 ml of Hank's balanced salt solution toremove noningested zymosan particles. The monolayers were then fixed andstained with Giemsa. The ingestion of zymosan particles by at least 300monocytes per monolayer was determined by-visual observation under a1000× light microscope.

Monocyte monolayers pretreated with buffer, 50 or 500 μg/ml of neutralsoluble glucan as described above were subsequently tested for theircapacity to ingest IgG coated sheep erythrocytes (E^(s) IgG). After an18 minute preincubation with the neutral soluble glucan, the monolayerswere incubated with 0.25 ml of 1×10⁷ /ml E^(s) IgG for 30 minutes at 37°C., washed three times with 50 ml of Hank's balanced salt solution,treated for 4 minutes with 0.84% NH₄ Cl to lyse noningested E^(s) IgG,and fixed and stained as described above. The percentages of monocytesingesting ≧1 and ≧3 E^(s) IgG were determined by counting at least 300monocytes per monolayer.

The percent inhibition of monocyte ingestion was determined bysubtracting the percentage of monocytes ingesting targets afterpretreatment with the neutral soluble glucan from the percentageingesting targets after pretreatment with buffer, dividing this numberby the percentage ingesting targets after pretreatment with buffer andmultiplying by 100. The data are expressed as the mean of twoexperiments and are reported in Table 5.

                  TABLE 5                                                         ______________________________________                                        Glucan-receptor Binding Capacity of                                           Distinct Conformations of Soluble β-glucans                              Test Material    Conc.    % Inhibition                                        ______________________________________                                        Buffer           --        0%                                                 Neutral soluble glucan                                                                          50 μg/ml                                                                           74%                                                                  500 μg/ml                                                                           86%                                                 Peak B            50 μg/ml                                                                           50%                                                                  500 μg/ml                                                                           56%                                                 ______________________________________                                    

Both β-glucan preparations tested above inhibited monocyte ingestion ofzymosan particles demonstrating their capacity to competitively bind tothe β-glucan receptor on human monocytes. Neutral soluble glucandemonstrated a higher receptor binding capacity than Peak B as indicatedby the greater level of inhibition achieved at both 50 μg/ml and 500μg/ml. This biological assay demonstrates that the neutral solubleglucan is a superior ligand for the β-glucan receptor.

Example 6

Lack of In Vitro Stimulation of IL-1β and TNFα From Human MononuclearCells

Venous blood was obtained from healthy male volunteers and mononuclearcells were fractionated by Ficoll-Hypaque centrifugation. Themononuclear cells were washed, resuspended in endotoxin-free RPMI-1640culture medium--ultrafiltered to remove endotoxins as describedelsewhere (Dinarello et al., 1987, J. Clin. Microbiol. 25:1233-8)--at aconcentration of 5×10⁶ cells/ml and were aliquoted into 96-wellmicrotiter plates (Endres et al., 1989, N.E. J. Med. 320:265-271). Thecells were then incubated with either 1 ng/ml endotoxin(lipopolysaccharide, E. coli 055:B5, Sigma, St. Louis), or 10 to 1000ng/ml β-glucan, at 37°0 C. for 24 hours in 5% CO₂ and then lysed bythree freeze-thaw cycles (Endres et al., 1989, N.E. J. Med.320:265-271). Synthesis of IL-1β and TNFα was determined by specificradioimmunoassays as described elsewhere (Lisi et al., 1987, Lym ph Res.6:229-244; Lonnemann et al., 1988, Lymph. Res. 7:75-84; Van der Meer etal., 1988, J. Leukocyte Biol. 43:216-223.

To determine if neutral soluble glucan could act as a priming agent forcytokine synthesis with endotoxin, a known cytokine stimulant,mononuclear cells were pre-incubated with 1, 10, and 1000 ng/ml of theneutral soluble glucan for 3 hours at 37° C. in 5% CO₂. The cells werewashed to remove neutral soluble glucan and were then incubated with 1ng/ml endotoxin as described above. IL-1 β and TNFα were determined asdescribed above.

The results are summarized in Table 6. Neutral soluble glucan used as astimulant at doses of 10-1000 ng/ml alone did not induce increasedlevels of IL-1 β or TNFα synthesis over the control buffer treatedcells. Endotoxin LPS, a known stimulant, resulted in significantlyincreased levels of both cytokines. In a second phase of this experimentneutral soluble glucan was tested for its ability to act as a primingagent for mononuclear cell cytokine synthesis. The cells from the samedonors were pre-incubated with three doses of neutral soluble glucan(10-1000 ng/ml) and were then exposed to endotoxin as a co-stimulant.Neutral soluble glucan did not result in any amplification of the IL-1βand TNFα levels compared to endotoxin alone.

                  TABLE 6                                                         ______________________________________                                        In Vitro IL-1β and TNFα Synthesis by                               Human Peripheral Blood Mononuclear Cells                                                            IL-1β                                                                            TNFα                                      Stimulant             (ng/ml).sup.1                                                                         (ng/ml).sup.1                                   ______________________________________                                        Cells only                <0.10   0.14                                        Neutral soluble glucan                                                                       10 ng/ml   0.13    0.16                                                       100 ng/ml  0.12    0.16                                                      1000 ng/ml  <0.10   0.14                                        LPS             1 ng/ml   2.62    2.22                                        LPS (1 ng/ml) +                                                                              10 ng/ml   2.62    2.25                                        Neutral soluble glucan                                                                       100 ng/ml  2.57    2.07                                                      1000 ng/ml  2.85    2.27                                        ______________________________________                                         .sup.1 Values are the mean of two donors.                                

Example 7

In Vitro Protection Against Infection in Rats

A sepsis model was developed in rats to characterize the efficacy ofβ-glucan in protecting an immunologically intact host against seriousinfections, such as those which commonly occur following abdominalsurgery. The rat model for intra-abdominal sepsis has been welldescribed in the scientific literature (Onderdonk et al., 1974, Infect.Immun., 10:1256-1259).

Groups of rats received neutral soluble glucan (100 μg/0.2 ml) or salinecontrol (0.2 ml) intramuscularly 24 hours and 4 hours prior toinfectious challenge. A defined polymicrobic infectious challenge (cecalinoculum) was placed into a gelatin capsule which was then surgicallyimplanted into the peritoneal cavity of anesthetized rats through ananterior midline incision. The early peritonitis from thisexperimentally induced infection was associated with the presence ofgram-negative organisms within the blood and peritoneal cavityculminating in mortality. The cecal inoculum contained an array offacultative species, such E. coli, as well as other obligate anaerobes(Streptococcus sp., Bacteroides sp., Clostridium perfringens,Clostridium ramosum, Peptostreptococcus magnus and productus, Proteusmirabilis). The animals were observed four times per day for the first48h and twice per day thereafter. The results are reported in Table 7.

                  TABLE 7                                                         ______________________________________                                        Effect of Neutral Soluble Glucan on Mortality                                 in a Rat Model for Intra-abdominal Sepsis                                     Group           Mortality (%)                                                                            P vs. Saline                                       ______________________________________                                        Saline          12/20 (60)                                                    Neutral soluble glucan                                                                         2/10 (10) <0.01                                              ______________________________________                                    

These results demonstrate that neutral soluble glucan which does notinduce IL-1β and TNFα protects rats from lethal bacterial challenge.

Example 8

Demonstration of Safety for Human Administration

A randomized, double-blind, placebo-controlled clinical trial wasconducted on healthy males to evaluate the safety of neutral solubleglucan (2.25 mg/kg) injected by intravenous infusion compared to aplacebo control. No adverse effects were observed. There was also noobserved elevation in IL-1, TNF, IL-6, IL-8 and GM-CSF. Singleintravenous administration of neutral soluble glucan resulted in anincrease in monocytes and neutrophils and in the killing activity ofthese cells proving that neutral soluble glucan retains the desirableimmunological activities in humans. See Tables 8, 9 and 10 below.However, as shown in FIGS. 5 and 6 no changes occurred in serum IL-1 andTNF and none of the patients experienced fever or inflammatoryreactions. The results are consistent with the in vitro data reported inthe earlier examples.

                  TABLE 8                                                         ______________________________________                                        Change In Absolute Neutrophil Counts (× 1000/μl)                     After Neutral Soluble Glucan Administration                                   Dose Level      B        Hour 8  Hour 12                                                                             Hour 24                                ______________________________________                                        Saline       Mean   4.06     4.34  4.31  3.43                                              SD     2.12     1.53  1.16  1.46                                              N      6        6     6     6                                    2.5 mg/kg    Mean   4.11     11.29*                                                                              8.18  5.32                                 Neutral Soluble Glucan                                                                     SD     1.15     4.39  3.80  1.75                                              N      6        6     6     6                                    ______________________________________                                         B = Baseline measurement                                                      *p < 0.01 with respect to baseline                                       

                  TABLE 9                                                         ______________________________________                                        Change in Monocyte Counts (× 1000/μl) After Glucan                   Administration                                                                Dose Level      B        Hour 8  Hour 12                                                                             Hour 24                                ______________________________________                                        Saline       Mean   0.33     0.44  0.59  0.33                                              SD     0.09     0.10  0.22  0.12                                              N      6        6     6     6                                    2.50 mg/kg   Mean   0.24      0.63*                                                                               0.67*                                                                              0.31                                 Neutral Soluble Glucan                                                                     SD     0.10     0.24  0.32  0.15                                              N      6        6     6     6                                    ______________________________________                                         B = Baseline measurement                                                      *p < 0.01 with respect to baseline                                       

                                      TABLE 10                                    __________________________________________________________________________    Ex Vivo Microbicidal Activity of Normal Volunteers                            Receiving Neutral Soluble Glucan Mean Change in % Killing.sup.1               Dose Level     Hour 3                                                                            Hour 6                                                                            Hour 24                                                                           Day 2                                                                             Day 3                                                                             Day 6                                      __________________________________________________________________________    Saline         0   0   0   0   0   0                                          2.5 mg/kg  Mean                                                                              42.86                                                                             32.33                                                                             20.90                                                                             48.96                                                                             39.22                                                                             31.17                                      Neutral Soluble Glucan                                                                   N   6   6   6   6   6   6                                                     p-Value                                                                           0.062                                                                             0.036                                                                             0.300                                                                             0.045                                                                             0.085                                                                             0.026                                      __________________________________________________________________________     .sup.1 Normalized with respect to the saline control                     

Example 9

Demonstration of Efficacy In Vivo as Human Anti-Infective

In this clinical study, the safety, tolerance, and potential efficacy ofthe neutral soluble β-glucan was evaluated in patients undergoing majorthoracoabdominal surgery with high risk of post-operative infection.Thirty-four males and females who underwent surgery received 0.5 mg/kgof the neutral soluble β-glucan preparation or saline placebo, given asan intravenous infusion of 50 to 200 ml over one hour. Patients receivedmultiple sequential doses of the neutral soluble βglucan or placebo at12 to 24 hours prior to surgery, 1 to 4 hours prior to surgery, 48 hourspost-surgery, and 96 hours post-surgery.

Hospitalization, infections, and usage of anti-infective medicationswere examined as potential clinical efficacy parameters. Compared topatients given saline placebo infusions, patients who received theneutral soluble β-glucan spent an average of five fewer days in thehospital (12.3±6.1 days versus 17.3±15.5 days) and three fewer days inthe Intensive Care Unit (0.1±0.4 versus 3.3±6.3 days; p<0.03, one-wayanalysis of variance).

The number of anti-infective medication prescriptions written per studyday following surgery was consistently higher for control patients thanfor β-glucan recipient patients. Control patients were prescribed anaverage of three times the number of anti-infective medications asβglucan recipients over the time period from surgery to discharge(p<0.005). During the Treatment and Post-Treatment Follow-up Phases, atotal of 22 culture-confirmed infections in 5 control patients and 8infections in 5 β-glucan ecipient patients were identified (p<0.002).

Neutrophils (PMNs) and monocytes/macrophages (MOs) ere purified fromblood samples obtained at Baseline, Day 1, and Day 5 and examined forbasal and phorbol myrisate acetate stimulated microbicidal activityagainst Staphylococcus aureus, Escherichia coli and Candida albicans.The neutral soluble β-glucan treatment generally increased the basal andphorbol-induced microbicidal activity of MOs and PMNS.

Example 10

Wound Healing Effects of Neutral Soluble Glucans

Wound healing studies were performed in a hairless mouse model havingfull thickness wounds with and without Staphylococcus aureus infection.Hairless SKH-1 inbred mice (6-8 weeks of age) were anesthetized withether and a midline 3 cm full thickness longitudinal incision was madewith a number 10 scalpel blade, producing a full thickness wound thatdid not penetrate the underlying fascia. Incisions were closed usingsteel clips placed at 1 cm intervals.

Formulations of neutral soluble glucan in phosphate buffered saline wereapplied 30 minutes following wounding and reapplied at 24 hour intervalsduring the seven day post-operative period. Two micrograms of neutralsoluble glucan/mouse per day was topically applied. Wounds were examineddaily and rank-ordered for effectiveness of formulation for enhancementof visual based wound healing. Wounds were scored for closure on a scaleof 0-5, with 5 indicating the most healing. In one group of miceinfected, the wound was treated with a culture of 10⁷ Staphylococcusaureus 30 minutes after wounding and 2 hrs prior to treatment with theneutral soluble glucan formulation.

Histological evaluation of the wound site of each test group was made.The dermis of the control group (untreated wound) was heavilyinfiltrated with both lymphocytes and monocytes/macrophages. However,re-epithelialization that occurred at the epidermal layer wasincomplete. The tissue section showed that the dermal tissue was weak,in that the tissue integrity was not maintained when it was sectioned.

The histology of the wounded tissue isolated from mice treated for threedays with phosphate buffered saline containing the neutral solubleglucan showed that there was a heavy infiltration of macrophages andlymphocytes. Tissue integrity was good.

When topically applied to a wound, a composition of neutral solubleglucan stimulated white blood cell entry and activity at the wound siteand accelerated wound healing within the dermal layer of the wound.Furthermore, the composition effectively eliminated infection producedby bacterial infection (S. aureus) and prevented the progression tosepsis. Untreated wounds progressed to sepsis.

Example 11

Stimulation of Platelet Proliferation by Neutral Soluble Glucan

The platelet proliferation stimulatory effect of the neutral solubleglucan was tested in an animal model system following either irradiationor administration of the chemotherapeutic agent cisplatin. Theseexperiments demonstrated the unexpected platelet stimulatory effect.

More specifically, saline or neutral soluble glucan prepared asdescribed in Example 1 was administered to groups of 10 mice as a singleIV bolus 20 hours prior to radiation exposure. Mice were bilaterallyexposed to a total-body irradiation of 7.5-Gy. Fourteen days afterirradiation the mice were sacrificed and whole blood samples wereanalyzed for peripheral blood counts. As shown in FIG. 7, the plateletcell count from neutral soluble glucan-treated mice was increased nearly3-fold relative to saline-treated control levels.

In addition to tests on irradiated mice, cisplatin-treated mice werealso tested for the effect of the neutral soluble glucan on platelethematopoiesis. Balb/c mice were injected intravenously with cisplatin ata dose of 9.3 mg/kg through the tail vein one hour before injectingeither saline or the neutral soluble glucan, prepared as described inExample 1, intramuscularly in a single dose of 0 (saline) or 2 mg/kg onDay 0. Platelet counts were determined before treatment (Day 0) and at2, 4, 6, 8, and 10 days post-treatment. The results of this experimentare shown in FIG. 8. Each data point represents the mean and standarderror of platelet counts from five mice. The statistically significantdifferences (p<0.05) between the saline and neutral soluble glucan (2mg/kg) are noted.

Biological Deposit

Saccharomyces cerevisiae strain R4 Ad was deposited on Aug. 20, 1992with the American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md., under the terms of the Budapest Treaty. The strain hasbeen assigned ATCC accession number 74181. Upon issuance of a patent,this deposit will be irrevocable.

Equivalents

Those skilled in the art will recognize or be able to ascertain, usingno more than routine experimentation, many equivalents to the specificmaterials and components described herein. Such equivalents are intendedto be encompassed in the scope of the following claims:

What is claimed is:
 1. An underivatized, aqueous soluble β (1-3)-glucan in a triple helix conformation, said glucan capable of enhancing immune response without stimulating production of biochemical mediators that cause inflammatory side effects, in vitro.
 2. The underivatized, aqueous soluble β(1-3)-glucan of claim 1 which, when incubated for greater than 3 hours at a concentration of about 1 μg/ml with a human peripheral blood mononuclear cell culture of about 5×10⁶ cells/ml, results in a less than 2-fold increase in interleukin-1 β and tumor necrosis factor-α synthesis over levels obtained following an otherwise identical incubation with a buffered solution lacking the β-glucan component.
 3. The underivatized, aqueous soluble β(1-3)-glucan of claim 1 which, when incubated for greater than 3 hours at a concentration of about 1 μg/ml with an endotoxin-stimulated human peripheral blood mononuclear cell culture of about 5×10⁶ cells/ml, results in a less than 2-fold increase in interleukin-1β and tumor necrosis factor-α synthesis over levels obtained with endotoxin stimulation alone.
 4. The β(1-3)-glucan preparation of claim 3 in which the human peripheral blood mononuclear cells are stimulated with Escherichia coli lipopolysaccharide endotoxin at a concentration of about 1 ng/ml.
 5. The β(1-3)-glucan of claim 1 wherein the β(1-3) glucan is derived from yeast.
 6. The β-glucan of claim 5 wherein the yeast is a strain of Saccharomyces cerevisiae.
 7. The β(1-3)-glucan of claim 6 wherein Saccharomyces cerevisiae is strain R4 (NRRL Y-15903) or strain R4 Ad (ATCC 74181).
 8. An underivatized, aqueous soluble β(1-3)-glucan consisting essentially of a molecular species which migrates as a single peak when analyzed by gel permeation chromatography, the molecular species being characterized by a triple helix conformation.
 9. A method for producing underivatized, aqueous soluble β(1-3)-glucan having a triple helix conformation, comprising:a) treating a suspension of insoluble β(1-3)-glucan with an organic acid to dissolve the organic acid-soluble portion of the β(1-3)-glucan; b) treating the organic acid-soluble β(1-3)-glucan with alkali to denature the native conformation of the soluble β(1-3)-glucan; c) neutralizing the solution containing the denatured soluble β(1-3)-glucan to re-anneal the soluble β(1-3)-glucan; and d) purifying the re-annealed soluble β(1-3)-glucan to obtain an underivatized, aqueous soluble β(1-3)-glucan having a triple helix conformation.
 10. The method of claim 9 wherein the insoluble β(1-3)-glucan is a whole glucan particle.
 11. The method of claim 9 wherein step a) is performed at a pH of from about 1 to about 5 and a temperature of from about 20° to about 100° C.
 12. The method of claim 9 wherein the organic acid is acetic acid or formic acid.
 13. The method of claim 9 wherein step (b) is performed at a pH of from about 7 to about 14 and a temperature of from about 40° to about 121° C.
 14. The method of claim 9 further comprising the step of purifying the denatured β(1-3)-glucan prior to step (c) to remove insoluble β(1-3)-glucans and aggregated soluble β(1-3)-glucans therefrom.
 15. The method of claim 14 wherein the purification prior to step (c) is performed using 1,000 to 100,000 dalton nominal molecular weight cut-off ultrafilters.
 16. The method of claim 9 wherein step (c) is performed at a pH of about 3.5 to 11.0 and at a temperature of from about 50° to 700° C.
 17. The method of claim 9 wherein step (d) is performed using a 30,000 to 70,000 nominal molecular weight cut-off ultrafilter and a 100,000 to 500,000 nominal molecular weight cut-off ultrafilter.
 18. The method of claim 9 wherein the β(1-3) glucan is derived from yeast.
 19. The method of claim 18 wherein the yeast is a strain of Saccharomyces cerevisiae.
 20. The method of claim 19 wherein Saccharomyces cerevisiae is strain R4 (NRRL Y-15903) or strain R4 Ad (ATCC 74181).
 21. A composition comprising an underivatized, aqueous soluble β(1-3)-glucan which is in a triple helix conformation, said glucan capable of enhancing immune response without stimulating production of biochemical mediators that cause inflammatory side effects, in vitro, the underivatized, aqueous soluble β(1-3)-glucan being solubilized in a physiologically acceptable vehicle.
 22. A composition of claim 21 which, when incubated for greater than 3 hours at a concentration of about 1 μg/ml with a human peripheral blood mononuclear cell culture of about 5×10⁶ cells/ml, results in a less than 2-fold increase in interleukin-1β and tumor necrosis factor-α synthesis over levels obtained following an otherwise identical incubation with a physiologically acceptable vehicle lacking the β-glucan component.
 23. The composition of claim 21 wherein the β(1-3) glucan is derived from yeast.
 24. The composition of claim 21 which enhances microbicidal activity of phagocytic cells.
 25. The composition of claim 21 which enhances hemopoietic production of monocytes and neutrophils.
 26. The composition of claim 21 wherein the physiologically acceptable vehicle is selected from the group consisting of water, sterile saline, phosphate buffered saline, isotonic saline and dextrose.
 27. The composition of claim 21 wherein the concentration of glucan in the physiologically acceptable vehicle is from about 0.5 to 100 mg/ml.
 28. The composition of claim 21 wherein the composition is in the form of a liquid, tablet, gel, ointment, lotion, capsule, powder, solution, emulsion or cream.
 29. The composition of claim 21 wherein the yeast is a strain of Saccharomyces cerevisiae.
 30. The composition of claim 29 wherein Saccharomyces cerevisiae is strain R4 (NRRL Y-15903) or strain R4 Ad (ATCC 74181). 